Ribozymes are RNA molecules that are capable of performing various biochemical reactions. The best characterized ribozymes are those that provide site-specific cleavage of the sugar-phosphate backbone in RNAs. Naturally occurring ribozymes generally cleave RNA targets in cis, whereas artificial ribozymes have been designed to cut substrate molecules in both cis and trans configuration (Cech, Curr. Opin. Struct. Biol., 2:605-609, 1992).
Ribozyme-mediated RNA cleavage has been extensively studied in vitro (Uhlenbeck, Nature, 328:596-600, 1987; Haseloff et al., Nature, 334:585-591, 1988; Hampel et al., Nucleic Acids Res., 18:299-304, 1990). Small ribozymes have been demonstrated to cleave substrate molecules in trans with high efficiency. However, in vivo experiments have revealed important fundamental differences between the test tube and living cells, and the ability to utilize ribozymes in vivo is far from its full potential (e.g., Sarver et al., Science, 247:1222-1225, 1990; Ojwang et al., Proc. Natl. Acad. Sci. USA, 89:10802-10805, 1992; Ferbeyre et al., J. Biol. Chem., 271:19318-19323, 1996). Despite many examples of ribozyme-dependent inhibition of gene expression in different organisms, better ribozymes and ribozyme strategies are needed for in vivo applications.
Factors that limit ribozyme activity in vivo have been partially characterized. They include difficulties in: (1) providing sufficiently high levels of ribozymes due often to metabolic instability; (2) predicting folding of the target and catalytic RNAs, which is essential as the catalytic and target RNAs interact through exposed complementary sequences; (3) achieving co-localization of the ribozymes and target RNAs in the cell; and (4) predicting, and thus avoiding, undesirable RNA-RNA and/or RNA-protein interactions that interfere with the desired function.